Method for preventing, treating, or ameliorating a microbial infection

ABSTRACT

A method for preventing, treating, or ameliorating a microbial infection can include administering thymoquinone or a pharmaceutical composition comprising thymoquinone to a patient in need thereof. The patient may be suffering from a microbial infection caused by gram-negative bacteria, gram-positive bacteria, or fungi. The microbial infection may be caused by gram negative bacteria. The gram-negative bacteria may include Acinetobacter baumannii. The gram-negative bacteria may include Pseudomonas aeruginosa. The microbial infection may be caused by antimicrobial sensitive Acinetobacter baumannii or antimicrobial resistant Acinetobacter baumannii.

BACKGROUND 1. Field

The disclosure of the present patent application relates toantimicrobial agents, and particularly to a thymoquinone antimicrobialagent and its use against drug-susceptible and drug-resistant microbialinfections.

2. Description of the Related Art

Pathogenic microorganisms such as bacteria and fungi cause infections inhumans and animals. Sometimes it is difficult to treat these infectionsusing available antimicrobial agents. In particular, the treatment oflife-threatening infections caused by resistant pathogenicmicroorganisms is a challenging task. In drug resistant cases,pathogenic microorganisms have reduced or lost their susceptibility toone or more antimicrobial agents. Antimicrobial resistance reduces theefficacy of antimicrobial drugs or renders the drug(s) ineffectiveagainst targeted microbe(s) at a standard dose. Treatment of infectionscaused by resistant microbes is difficult, complicated and expensive. Insuch cases, patients' suffering is prolonged and chances of mortalityare increased. Multidrug resistance limits the therapeutic optionsavailable for treatment of infections and forces healthcare providers touse comparatively higher doses or more toxic and/or expensiveantimicrobial agents.

Gram-negative infections are typically due to common pathogens such asAcinetobacter, Pseudomonas aeruginosa, and the Enterobacteriaceaefamily. Acinetobacter baumannii infections are typicallyhospital-derived infections.

Thus, a method for treating microbial infections solving theaforementioned problems is desired.

SUMMARY

A method for preventing, treating, or ameliorating a microbial infectioncan include administering thymoquinone or a pharmaceutical compositioncomprising thymoquinone to a patient in need thereof. The microbialinfection may be caused by gram-negative bacteria, gram-positivebacteria, or fungi. The gram-negative bacteria may include Acinetobacterbaumannii. The gram-negative bacteria may include Pseudomonasaeruginosa. The microbial infection may be caused by antimicrobialsensitive Acinetobacter baumannii or antimicrobial resistantAcinetobacter baumannii. The pharmaceutical composition may beformulated as a micro-emulsion.

These and other features of the present disclosure will become readilyapparent upon further review of the following specification anddrawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A method for preventing, treating, or ameliorating a microbial infectioncan include administering thymoquinone or a pharmaceutical compositioncomprising thymoquinone to a patient in need thereof. The patient mayinclude a human or an animal. The microbial infection may be caused bygram-negative bacteria, gram-positive bacteria, or fungi. The microbialinfection may be caused by gram negative bacteria. The gram-negativebacteria may include Acinetobacter baumannii. The gram-negative bacteriamay include Pseudomonas aeruginosa. The microbial infection may becaused by antimicrobial sensitive Acinetobacter baumannii orantimicrobial resistant Acinetobacter baumannii.

As used herein, the term “about” when modifying a numerical value shallmean within 10% of the modified numerical value.

Thymoquinone (2-isopropyl-5-methylbenzo-1, 4-quinone) is a bioactivemolecule. Thymoquinone has been reported to have anti-oxidant activity,anti-inflammatory activity, and anti-cancer activity. Thymoquinone hasalso demonstrated protective effects on several organs against oxidativedamage.

A pharmaceutical composition comprising thymoquinone can includethymoquinone and at least one pharmaceutically acceptable excipients.Pharmaceutically acceptable excipients can be selected from carriers,diluents, stabilizers, complexing agents, buffers, binders, emulsifiers,surfactants, solubilizers, thickeners, suspending agents, hydrophobicointment base, gel forming polymers, lubricants, colors, flavors, andpreservatives. In an embodiment, the pharmaceutical compositioncomprising thymoquinone is formulated as a microemulsion includingthymoquinone, an oil, a surfactant, and water. In an embodiment, thepharmaceutical composition includes from about 0.01% to about 1.0% w/vthymoquinone, from about 2% to about 6% by volume oil, from about 25% toabout 40% by volume surfactant, and a remainder of the pharmaceuticalcomposition includes water.

The pharmaceutical composition may include at least one supplementaryantimicrobial agent. The thymoquinone and the at least one supplementalantimicrobial agent can be in intimate contact in the pharmaceuticalcomposition. The thymoquinone and the at least one supplementalantimicrobial agent in the pharmaceutical composition may be separatedby a barrier. The thymoquinone and the at least one additionalsupplemental antimicrobial agent can be separately formulated, butsimultaneously used.

The supplemental antimicrobial agent can include at least one of thefollowing: aminoglycosides (Amikacin, Gentamicin, Kanamycin, Neomycin,Netilmicin, Tobramycin, Paromomycin, Streptomycin), Ansamycins(Geldanamycin, Rifaximin), Carbapenems (Ertapenem, Doripenem,Imipenem/Cilastatin, Meropenem), Cephalosporins (Cefadroxil, Cefazolin,Cefalexin, Cefaclor, Cefprozil, Cefuroxime, Cefixime, Cefdinir,Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime,Ceftibuten, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole)Glycopeptides (Teicoplanin, Vancomycin, Telavancin, Dalbavancin,Oritavancin) Lincosamides (Clindamycin, Lincomycin) Lipopeptide(Daptomycin) Macrolides (Azithromycin, Clarithromycin, Erythromycin,Roxithromycin, Telithromycin, Spiramycin) Monobactams (Aztreonam)Nitrofurans (Furazolidone, Nitrofurantoin) Oxazolidinones (Linezolid,Posizolid, Radezolid, Torezolid) Penicillins (Amoxicillin, Ampicillin,Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin,Nafcillin, Oxacillin, Piperacillin) Penicillin combinations(Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,Ticarcillin/clavulanate) Polypeptides (Bacitracin, Colistin, PolymyxinB) Quinolones/Fluoroquinolones (Ciprofloxacin, Enoxacin, Gatifloxacin,Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin,Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin,Sparfloxacin, Temafloxacin) Sulfonamides (Mafenide, Sulfacetamide,Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole,Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,Co-trimoxazole, Sulfonamidochrysoidine) Tetracyclines (Demeclocycline,Doxycycline, Metacycline, Minocycline, Oxytetracycline, Tetracycline)Anti-mycobacterials (Capreomycin, Cycloserine, Ethambutol, Ethionamide,Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine,Streptomycin) other antibiotics (Arsphenamine, Chloramphenicol,Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Thiamphenicol,Tigecycline), essential oils and their active constituents (Clove oil,Eugenol, Cinnamaldehyde), and myrrh (Commiphora myrrha) and its activeingredients.

The methods described herein may be used to prevent, treat, orameliorate microbial infections caused by gram-positive bacteria, gramnegative bacteria, or fungi. The methods may be used to prevent, treat,or ameliorate antimicrobial sensitive as well as antimicrobial resistantmicroorganisms.

The pathogenic gram-positive bacteria may include Staphylococcus (e.g.,S. epidermis and S. aureus); Corynebacterium (e.g., C. pyogenes and C.pseudotuberculosis); Micrococcus; Streptococcus (e.g., S. pyogenes, S.equis, S. zooepidemicus, S. equisimilis, S. pneumoniae and S.agalactiae); Erysipelothrix (e.g, E. rhusiopathiae); Bacillus (e.g., B.anthracis); Listeria (e.g., L. monocytogenes); Clostridium (C.perfringens); and Mycobacterium (M. tuberculosis and M. leprae). Animalor human subjects infected with these pathogens may be detected byestablished methods.

The pathogenic gram negative bacteria infection may includeEnterobacteriaceae (Escherichia, e.g., E. coli; Citrobacter;Enterobacter; Shigella; Salmonella; Edwardsiella; Hafnia; Klebsiella,e.g., K. pneumoniae; Morganella; Proteus; Providencia; Serratia;Yersinia); Pseudomonadaceae (Pseudomonas, especially P. aeruginosa,Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas, Comamonas),Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella,Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella,Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae (Treponema andBorrelia), Bacteroidaceae (Bacteroides, Fusobacterium, Prevotella,Porphyromonas), Leptospiraceae, Campylobacter, Helicobacter, Spirillum,Streptobacillus, Acinetobacter, especially Acinetobacter baumanii.Animal or human subjects infected with these pathogens may be detectedby established methods.

The pathogenic fungi may include Candida (e.g., C. albicans); Malassezia(e.g., M dermatis); Coccidioides (e.g., C. immitis); Penicillium (e.g.,P. marneffei); and Pneumocystis (e.g., P. carinii); Hyphomyces (e.g., H.destruens); Cryptococcus (e.g., C. neoformans); Aspergillus (e.g., A.fumigatus); Histoplasma (e.g., H. capsulatum); Blastomyces (e.g., B.dermatiditis); Rhinosporidium; Sporothrix; and dermatophytes. Animal orhuman subjects infected with these pathogens may be detected byestablished methods.

In an embodiment the pharmaceutical composition may be administered to ahuman or animal patient in a suitable form. A suitable form for topicaladministration (skin and mucous membrane) may include a topicalointment, cream, micro-emulsion, nano-emulsion, spray, lotion, oil,powder, liniment, solution, or a gel. A suitable form for oraladministration may include a tablet, capsule, pellet, granules,solution, lozenge, suspensions, emulsions, micro-emulsion, or anano-emulsion. The pharmaceutical composition can also be formulated asinjectable solution, powder ready for reconstitution for injection,inhalant, or a suppository.

In an embodiment, the pharmaceutical composition may be formulated as amicroemulsion. The microemulsion can include thymoquinone, an oil, atleast one of a surfactant and a co-surfactant, and water. The oil can beselected from fixed oils and essential oils.

In an embodiment the thymoquinone antimicrobial agent may be protectedfrom light and packaged in suitable light resistant container. Thethymoquinone antimicrobial agent may be packaged in aluminum tubes,light resistant plastic tubes, light resistant bottles or jar likecontainers, or in light resistant blister packaging.

In an embodiment, the pharmaceutical composition may be deliveredtopically to the patient via a dressing comprising thymoquinone. Thedressing may also comprise an additional antimicrobial agent along withthymoquinone. Suitable dressings may include bandages, gauzes, tissues,films, gels, or foams.

In an embodiment, the pharmaceutical composition may be provided forsystemic use. For systemic use, the pharmaceutical composition can beadministered orally, parenterally (intravenous, intramuscular, orsubcutaneous injections), or by the nasal route (through solution orinhalator or spray).

In an embodiment, the pharmaceutical composition may be provided fortopical use. For topical use, the pharmaceutical composition may beadministered on the skin or on a mucous membrane (e.g., buccal mucosa,eyes and ears, rectal mucosa, vaginal mucosa) of the patient.

For prevention, treatment or amelioration of infection, the thymoquinoneantimicrobial agent may be administered or applied at an appropriatefrequency. The thymoquinone antimicrobial agent may be administered orapplied at least once a day, twice a day, or thrice a day. Thethymoquinone antimicrobial agent may be administered or applied at anappropriate frequency for at least 3 days.

For systemic or topical treatment of infection, the amount ofthymoquinone administered will depend on multiple factors including ageand weight of the subject, severity and size of the wound, the numbersand types of additional active antimicrobial agents contained in thecomposition administered, and the ratio of active antimicrobial agentsin the composition.

The thymoquinone antimicrobial agent may be administered upon detectingemerging antibiotic resistance.

The thymoquinone antimicrobial agent may be used for the treatment ofskin infections, such as burn and wound infections, especially thosecaused by Acinetobacter baumannii.

The thymoquinone antimicrobial agent may be used for the treatment ofinfections in animals. These animal infections may include skininfections, such as burn and wound infections, especially those causedby Acinetobacter baumannii.

The following examples illustrate the present teachings:

EXAMPLES Example 1 Method of Making a Micro-Emulsion Composition withThymoquinone and Clove Oil Microemulsion 1

A pharmaceutical composition was prepared including thymoquinone (0.1%w/v), Clove Oil (3% v/v), Tween-20 (30% v/v) and Water (q.s. up to100%). Briefly, thymoquinone was dissolved in clove oil to form a firstmixture and then tween-20 was added to form a second mixture. The secondmixture was vortexed for 1 to 2 minutes and then sonicated for 2-3minutes in a bath sonicator. After sonication, water was added dropwiseto the second mixture, while continuously vortexing to provide amicroemulsion. The resulting pharmaceutical composition comprising amicro-emulsion of thymoquinone, clove oil, tween-20 and water wasfurther sonicated to remove air bubbles.

Example 2 Method of Making a Control Micro-Emulsion Composition withClove Oil Microemulsion 2

A pharmaceutical composition was prepared containing clove Oil (3% v/v),Tween-20 (30% v/v), and water (q.s. up to 100%). Briefly, clove oil andtween 20 were mixed together to form a mixture. The mixture was vortexedfor 2 to 3 minutes and then sonicated for 2-3 minutes in a bathsonicator. After sonication, water was added dropwise to the mixture,while continuously vortexing to provide a microemulsion. The resultingpharmaceutical composition comprising a micro-emulsion of clove oil,tween-20 and water was further sonicated to remove air bubbles.

Example 3 Determination of Antimicrobial Susceptibility (Acinetobacterbaumanii Strains)

A total of 33 non-duplicate, non-consecutive clinical isolates ofMulti-Drug Resistant (MDR) Acinetobacter baumannii were collected fromburn wards (“AB1-AB33”). Acinetobacter baumannii ATCC BAA 747 andPseudomonas aeruginosa ATCC 27853 were used as control strains. Theisolates were stored at −80° C. in trypticase soy broth containing 20%glycerol.

Antimicrobial susceptibility tests of the collection of isolates wereconducted using the disc diffusion method. The experiment was performedaccording to the recommendations and guidelines of Clinical LaboratoryStandard Institutes (CLSI M100-S24; 2014). Clinical and LaboratoryStandards Institute (CLSI), “Performance standards for antimicrobialsusceptibility testing; twenty-fourth informational supplement,”Document M100-S24, Clinical and Laboratory Standards Institute (CLSI),Wayne, Pa., USA, 2014.

A panel of commercial antibiotic discs (Amoxicillin (AMX),Amoxicillin/clavulanic acid (AMX/CLA), Piperacillin (PIP),Piperacillin/Tazobactam (PIP/TAZ), Ceftazidime (CAZ), Cefotaxime (CTX),Cefepime (FEP), Cefoxitin (FOX), Aztreonam (ATM), Imipenem (IP),Gentamicin (GEN), Amikacin (AK), Neomycin (NEO), Levofloxacin (LEV),Chloramphenicol (CHL), Tetracycline (TET), sulfamethoxazole/trimethoprim(SUL/TRI) was used for susceptibility testing. All commercial antibioticdiscs were purchased from BBL (Becton Dickinson, USA).

Forty grams of Mueller-Hinton Agar (MHA) were suspended in 1 Literdistilled water and then boiled to dissolve the powder completely. Themedium was sterilized by autoclaving at 121° C. for 15 min. Afterautoclaving, the medium was cooled to 45° C. and 25 mL of molten agarmedia was poured into sterile Petri dishes (90 mm diameter) to give adepth of about 4 mm. The surface of the agar was dried to remove excessmoisture before use. The plates were stored at 4-8° C. in sealed plasticbags if not used immediately.

The inoculum suspension was prepared by selecting three to fivemorphologically similar colonies from overnight growth (16-24 h ofincubation) on blood agar medium. The colonies were suspended in sterilenormal saline to give the density of a McFarland 0.5 standard, which isapproximately equivalent to 1-2×10⁸ CFU/mL. The suspension density wasmeasured using a UV spectrophotometer calibrated with a 0.5 McFarlandstandard. The density of the suspension was adjusted to McFarland 0.5 byaddition of saline or more microorganisms. The adjusted inoculumsuspension was used within 60 min of preparation.

A sterile cotton swab was dipped into the bacterial suspension and theexcess fluid was removed by turning the swab against the inside wall ofthe tube to avoid over-inoculation of plates. The inoculum was spreadevenly over the entire surface of the agar plate by swabbing in threedirections.

The predetermined panel of antimicrobial disks was applied firmly on theinoculated Mueller-Hinton agar surface within 15 min of inoculation ofthe plates by a disc dispenser device. A maximum of six disks wereaccommodated on a 90-mm circular plate.

Within 15 min of application of antimicrobial disks, the plates wereinverted and incubated aerobically at 35-37° C. for 18-20 h.

After incubation, inhibition zones were measured at the point where noobvious growth is detected by the unaided eye when the plate is heldabout 30 cm from the eye. Inhibition zone diameters were measured to thenearest millimeter with a ruler. The plates were read from the back ofthe plate with reflected light against a dark background. Zone diameterswere interpreted and categorized as susceptible “S”, intermediate “I”,or resistant “R” according to the CLSI clinical breakpoint tables (seeTable 1 and Table 2). “NZ” indicated that no zone of inhibition wasobserved.

TABLE 1 Antibiotic Susceptibility Testing Against AcinetobacterBaumannii Isolates (Inhibition zone in mm) AMX AMX/CLA PIP PIP/TAZ CAZCTX FEP FOX AB1 NZ R NZ R 21 S 22 S 20 S NZ R 20 S NZ R AB2 NZ R NZ R 20I 22 S 17 I NZ R 16 I NZ R AB3 NZ R NZ R 32 S 35 S 26 S 15 I 18 S NZ RAB4 NZ R NZ R 21 S 22 S 21 S  8 R 14 R NZ R AB5 NZ R NZ R NZ R NZ R  9 RNZ R  7 R NZ R AB6 NZ R NZ R 18 I 18 I 28 S 17 I 21 S NZ R AB7 NZ R NZ RNZ R NZ R NZ R NZ R NZ R NZ R AB8 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZR AB9 NZ R NZ R 24 S 25 S 21 S 11 R 22 S NZ R AB10 NZ R NZ R NZ R NZ RNZ R NZ R  7 R NZ R AB11 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZ R AB12 NZR NZ R NZ R NZ R NZ R NZ R NZ R NZ R AB13 NZ R NZ R NZ R NZ R NZ R NZ RNZ R NZ R AB14 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZ R AB15 NZ R NZ R NZR NZ R NZ R NZ R  8 R NZ R AB16 NZ R NZ R 16 R 25 S 32 S 32 S 35 S 22 SAB17 NZ R NZ R 15 R 15 R 12 R NZ R 11 R NZ R AB18 NZ R NZ R  8 R  8 R NZR NZ R NZ R NZ R AB19 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZ R AB20 NZ RNZ R NZ R NZ R NZ R NZ R NZ R NZ R AB21 NZ R NZ R NZ R NZ R NZ R NZ R NZR NZ R AB22 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZ R AB23 NZ R NZ R NZ R10 R NZ R NZ R NZ R NZ R AB24 NZ R NZ R NZ R  9 R NZ R NZ R  7 R NZ RAB25 NZ R NZ R 10 R 12 R 17 I 16 I 10 R NZ R AB26 NZ R NZ R NZ R  9 R 15I NZ R 14 R NZ R AB27 NZ R 7 R  8 R 12 R  8 R NZ R  8 R NZ R AB28 9 R 8R  7 R  9 R  8 R NZ R NZ R NZ R AB29 NZ R NZ R NZ R NZ R NZ R NZ R NZ RNZ R AB30 NZ R NZ R  8 R 10 R NZ R NZ R NZ R NZ R AB31 8 R 8 R  8 R  9 R 8 R  8 R NZ R  9 R AB32 8 R 8 R  8 R 10 R  8 R  8 R NZ R NZ R AB33 NZ RNZ R 22 S 23 S 22 S 21 I 18 S NZ R Break Points Sensitive ≥17 ≥18 ≥21≥21 ≥18 ≥23 ≥18 ≥18 Intermediate 14-16 14-17 18-20 18-20 15-17 15-2215-17 15-17 Resistant ≤13 ≤13 ≤17 ≤17 ≤14 ≤14 ≤14 ≤14

TABLE 2 Antibiotic Susceptibility Testing Against AcinetobacterBaumannii Isolates (Inhibition zone in mm) ATM IP GEN AK NEO TET CHLSUL/TRI LEV AB1 NZ R NZ R 24 S 24 S 23 S NZ R NZ R NZ R 30 S AB2 NZ R NZR 20 S 20 S 20 S 22 S 24 S 23 S 30 S AB3 NZ R NZ R 24 S 26 S 22 S 22 S26 S 23 S 29 S AB4 NZ R NZ R 22 S 21 S 27 S 21 S 23 S 20 S 30 S AB5 NZ R 8 R 15 S  7 R 10 R NZ R  8 R NZ R 19 S AB6 20 S 12 R 26 S 30 S 14 I NZR NZ R NZ R 21 S AB7 NZ R NZ R NZ R NZ R 12 R 21 S NZ R NZ R 14 I AB8 NZR  7 R NZ R 12 R 11 R 26 S  7 R NZ R 15 I AB9 NZ R NZ R 21 S 22 S 20 S23 S 24 S 25 S 28 S AB10 13 R NZ R NZ R NZ R NZ R NZ R NZ R  8 R 14 IAB11 NZ R NZ R NZ R NZ R NZ R NZ R NZ R NZ R 11 R AB12 NZ R 11 R 12 R 21S 15 S NZ R NZ R NZ R 10 R AB13 NZ R  9 R NZ R 22 S 9 R NZ R NZ R NZ R 8 R AB14 NZ R 15 R NZ R NZ R NZ R 24 S NZ R NZ R 11 R AB15 11 R NZ R 20S 13 R 13 I 19 S 10 R NZ R 15 I AB16 33 S 34 S 18 S 19 S NZ R NZ R 30 S26 S NZ R AB17 15 R 15 R 24 S 22 S 20 S 21 S 13 I NZ R 11 R AB18 10 R NZR 17 S 14 R 15 S 20 S  8 R NZ R 13 R AB19  8 R NZ R 20 S 14 R 13 I 17 S 8 R NZ R 11 R AB20 NZ R 15 R NZ R NZ R NZ R 28 S NZ R NZ R 13 R AB21 7R NZ R 17 S 11 R 13 I 19 S NZ R NZ R 14 I AB22 NZ R 15 R NZ R NZ R  7 R20 S NZ R NZ R 14 I AB23 NZ R 20 I NZ R NZ R 10 R 23 S NZ R NZ R 11 RAB24 NZ R NZ R 19 S 14 R 14 I 19 S  8 R NZ R 12 R AB25 13 R 23 S 19 S NZR  8 R 25 S NZ R 21 S 14 I AB26 12 R 22 S NZ R 18 S 20 S NZ R 25 S NZ RNZ R AB27 NZ R  9 R  8 R  9 R  9 R  8 R NZ R NZ R 8 R AB28 NZ R  8 R 20S 15 I 15 S 20 S 14 I NZ R 15 I AB29 NZ R  9 R 20 S 15 I 15 S 19 S 12 RNZ R 15 I AB30  9 R  9 R 20 S 15 I 15 S 20 S 11 R NZ R 15 I AB31 NZ R  9R  8 R NZ R  8 R NZ R  8 R NZ R 10 R AB32  8 R 18 R NZ R NZ R 10 R 28 S 9 R NZ R 18 S AB33  9 R NZ R 20 S 25 S 20 S 24 S 18 S 24 S 30 S BreakPoint Sensitive ≥22 ≥22 ≥15 ≥17 ≥15 ≥15 ≥18 ≥16 ≥17 Intermediate 16-2119-21 13-14 15-16 13-14 12-14 13-17 11-15 14-16 Resistant ≤15 ≤18 ≤12≤14 ≤12 ≤11 ≤12 ≤10 ≤13

Example 4 Thymoquinone MIC Determination

Minimum Inhibitory Concentration (MIC) is the lowest concentration of anantimicrobial agent that prevents visible growth of a microorganism inan agar or broth dilution susceptibility test. MIC of thymoquinone wasdetermined by the broth dilution method against 33 MDR Acinetobacterbaumannii strains and standard sensitive culture collection strain ofAcinetobacter baumannii and Pseudomonas aeruginosa. The MIC ofgentamicin was deter lined by broth dilution method against six MDRAcinetobacter baumannii strains and standard sensitive culturecollection strains of Acinetobacter baumannii and Pseudomonasaeruginosa. MIC testing was performed according to the recommendationsand guidelines of Clinical Laboratory Standard Institutes (CLSI M07-A9;2012). Clinical and Laboratory Standards Institute (CLSI), “Methods forDilution Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically; Approved Standard,” Document M07-A9, Clinical andLaboratory Standards Institute (CLSI), Wayne, Pa., USA, 2012.

Twenty-one grams of Mueller-Hinton Broth (MHB) was suspended in 1 Literdistilled water and then boiled to dissolve the powder completely. Themedium was sterilized by autoclaving at 121° C. for 15 min. Afterautoclaving, the medium was cooled to room temperature before use orstored in the fridge until used.

Thymoquinone was weighed and dissolved in Dimethyl sulfoxide (DMSO) toproduce a thymoquinone stock solution with a concentration of 5,120μg/ml. Gentamicin sulfate was weighed and dissolved in water to producea gentamicin sulfate stock solution with a concentration of 5,120 μg/ml,equivalent to gentamicin base.

Dilutions of thymoquinone and gentamicin sulfate stock solutions wereprepared volumetrically in the MHB as follows:

One mL of original stock solution (5,120 μg/ml) was added to 9 mlsterile MHB to give the working first dilution (512 μg/mL). 12 sterile 7mL PJ tubes were arranged in a rack in a row. Each tube contained one mLsterile MHB. Ten tubes were labeled as 128 μg/mL, 64 μg/mL, 32 μg/mL, 16μg/mL, 8 μg/mL, 4 μg/mL, 2 μg/mL, 1 μg/mL, 0.5 μg/mL and 0.25 μg/mL. Theeleventh and twelfth tubes were labeled as positive and negativecontrol, respectively.

The working dilution (512 μg/mL) was serially diluted to give two-folddilutions. One mL of the working dilution (512 μg/mL) was dispensed inthe first tube (labeled as 128 μg/mL) to give a concentration of 256 μgdrug/mL MHB. One mL of the first tube was added to the second tube(labeled as 64 μg/mL) to give a concentration of 128 μg drug/mL MHB. OnemL of the second tube was transferred to the third tube (labeled as 32μg/mL) to give a concentration of 64 μg drug/mL MHB. One mL of the thirdtube was added to the fourth tube (labeled as 16 μg/mL) to give aconcentration of 32 μg drug/mL MHB. One mL of the fourth tube wasdispensed in the fifth tube (labeled as 8 μg/mL) to give a concentrationof 16 μg drug/mL MHB. One mL of the fifth tube was dispensed in thesixth tube (labeled as 4 μg/mL) to give a concentration of 8 μg drug/mLMHB. One mL of the sixth tube was added to the seventh tube (labeled as2 μg/mL) to give a concentration of 4 μg drug/mL MHB. One mL of theseventh tube was added to the eighth tube (labeled as 1 μg/mL) to give aconcentration of 2 μg drug/mL MHB. One mL of the eighth tube wastransferred to the ninth tube (labeled as 0.5 μg/mL) to give aconcentration of 1 μg drug/mL MHB. One mL of the ninth tube wastransferred to the tenth tube (labeled as 0.25 μg/mL) to give aconcentration of 0.5 μg drug/mL MHB. The contents were mixed thoroughlyby pipetting up and down three times. One mL of the content of the tenthtube was discarded.

The inoculum suspensions (at 1-2×10⁸ CFU/mL) were diluted 1:100 in MHBto obtain 1×10⁶ CFU/mL. One mL of the diluted inoculum suspension (1×10⁶CFU/mL) was transferred to each tube except tube 12. This resulted in1:2 dilutions of each drug concentration and a 1:2 dilution of theinoculums (to 5×10⁵ CFU/mL).

All inoculated tubes were incubated for 16-20 hours at 37° C.aerobically. After this incubation period, MIC was determined manuallyby observing the lowest concentration of antimicrobial agent showing novisible growth (turbidity). The MIC of thymoquinone againstAcinetobacter baumannii isolates and standard strain of Acinetobacterbaumannii ATCC BAA 747 is presented in Table 3. The MIC of gentamicinagainst Acinetobacter baumannii isolates and standard strain ofAcinetobacter baumannii ATCC BAA 747 is presented in Table 4. The MIC ofthymoquinone and gentamicin against Pseudomonas aeruginosa ATCC 27853was found to be 64 μg/ml and ≤2 μg/ml, respectively.

TABLE 3 MIC of Thymoquinone Against Acinetobacter baumannii Isolates(μg/ml) Isolate MIC Isolate MIC Isolate MIC Isolate MIC AB1 32 AB10 16AB19 64 AB28 64 AB2 32 AB11 32 AB20 64 AB29 64 AB3 32 AB12 32 AB21 32AB30 32 AB4 32 AB13 32 AB22 64 AB31 32 AB5 32 AB14 32 AB23 32 AB32 32AB6 64 AB15 32 AB24 32 AB33 32 AB7 32 AB16 64 AB25 32 A.b. ATCC BAA 74732 AB8 32 AB17 64 AB26 64 P a. ATCC 27853 64 AB9 32 AB18 32 AB27 64

TABLE 4 MIC of Gentamicin Sulfate Against Acinetobacter baumanniiIsolates (μg/ml) Isolates MIC AB7 32 AB8 64 AB10 32 AB11 32 AB12 32 AB1332 A. baumannii ATCC BAA 747 1 P. aeruginosa ATCC 27853 ≤2

Example 5 Determination of MIC of the Micro-Emulsion of Example 1

Nine sterile 7 mL PJ tubes were arranged in a rack in a row. Each tubecontained one mL sterile MHB. Seven tubes were labeled as 7.847 mg+256μg/mL, 3.923 mg+128 μg/mL, 1.961 mg+64 μg/mL, 0.980 mg+32 μg/mL, 0.490mg+16 μg/mL, 0.245 mg+8 μg/mL and 0.122 mg+4 μg/mL. The eighth and ninthtubes were labeled as positive and negative controls, respectively.

The micro-emulsion made according to Example 1 was serially diluted togive two-fold dilutions as follows: one mL of the micro-emulsion ofExample 1 was dispensed in the first tube (labeled as 7.847 mg+256μg/mL) to give a concentration of 15.69 mg+512 μg drug/mL MHB. One mL ofthe first tube was added to the second tube (labeled as 3.923 mg+128μg/mL) to give a concentration of 7.847 mg+256 μg drug/mL MHB. One mL ofthe second tube was transferred to the third tube (labeled as 1.961mg+64 μg/mL) to give a concentration of 3.923 mg+128 drug/mL MHB. One mLof the third tube was added to the fourth tube (labeled as 0.980 mg+32μg/mL) to give a concentration of 1.961 mg+64 drug/mL MHB. One mL of thefourth tube was dispensed in the fifth tube (labeled as 0.490 mg+16μg/mL) to give a concentration of 0.980 mg+32 drug/mL MHB. One mL of thefifth tube was dispensed in the sixth tube (labeled as 0.245 mg+8 μg/mL)to give a concentration of 0.490 mg+16 μg drug/mL MHB. One mL of thesixth tube was added to the seventh tube (labeled as 0.122 mg+4 μg/mL)to give a concentration of 0.245 mg+8 μg drug/mL MHB. One mL of thecontent of the seventh tube was discarded. Inoculum suspensions (at1-2×10⁸ CFU/mL) were diluted 1:100 in MHB to obtain 1×10⁶ CFU/mL. One mLof the diluted inoculum suspension (1×10⁶ CFU/mL) was transferred toeach tube except tube number nine. This resulted in a 1:2 dilutions ofeach drug concentration and a 1:2 dilution of the inoculums (5×10⁵CFU/mL).

Another nine sterile 7 mL PJ tubes were arranged in a rack in a row.Each tube contained one mL sterile MHB. Seven tubes were labeled as7.847 mg/mL, 3.923 mg/mL, 1.961 mg/mL, 0.980 mg/mL, 0.490 mg/mL, 0.245mg/mL and 0.122 mg/mL. The eighth and ninth tubes were labeled aspositive and negative control, respectively.

The micro-emulsion of Example 2 was serially diluted to give two-folddilutions as follows: one mL of the micro-emulsion of Example 2 wasdispensed in the first tube (labeled as 7.847 mg/mL) to give aconcentration of 15.69 mg drug/mL MHB. One mL of the first tube wasadded to the second tube (labeled as 3.923 mg/mL) to give aconcentration of 7.847 mg drug/mL MHB. One mL of the second tube wastransferred to the third tube (labeled as 1.961 mg/mL) to give aconcentration of 3.923 mg drug/mL MHB. One mL of the third tube wasadded to the fourth tube (labeled as 0.980 mg/mL) to give aconcentration of 1.961 mg drug/mL MHB. One mL of the fourth tube wasdispensed in the fifth tube (labeled as 0.490 mg/mL) to give aconcentration of 0.980 mg drug/mL MHB. One mL of the fifth tube wasdispensed in the sixth tube (labeled as 0.245 mg/mL) to give aconcentration of 0.490 mg drug/mL MHB. One mL of the sixth tube wasadded to the seventh tube (labeled as 0.122 mg/mL) to give aconcentration of 0.245 mg drug/mL MHB. One mL of the content of seventhtube was discarded. Inoculum suspensions (at 1-2×10⁸ CFU/mL) werediluted 1:100 in MHB to obtain 1×10⁶ CFU/mL. One mL of the dilutedinoculum suspension (1×10⁶ CFU/mL) was transferred to each tube excepttube number nine. This resulted in a 1:2 dilution of each drugconcentration and a 1:2 dilution of the inoculums (to 5×10⁵ CFU/mL).

All inoculated tubes were incubated for 16-20 hours at 37° C.aerobically. After this incubation period, MIC was determined manuallyby observing the lowest concentration of antimicrobial agent showing novisible growth (turbidity). The MIC of the micro-emulsion of Example 1and the micro-emulsion of Example 2 against Acinetobacter baumanniiisolates is presented in Table 5.

TABLE 5 MIC of Example 1, Example 2, and Thymoquinone Against MultidrugResistant Acinetobacter baumannii Isolates (μg/ml) Isolate 121 Isolate127 Isolate 129 Example 1 16 ± 490.4 16 ± 490.4 16 ± 490.4 Example 21961.8 980.9 980.9 Thymoquinone 64 32 32

It is to be understood that a method for preventing, treating, orameliorating microbial infections is not limited to the specificembodiments described above, but encompasses any and all embodimentswithin the scope of the generic language of the following claims enabledby the embodiments described herein, or otherwise shown in the drawingsor described above in terms sufficient to enable one of ordinary skillin the art to make and use the claimed subject matter.

1-5. (canceled)
 6. A pharmaceutical composition, comprising from about0.01% to about 1.0% w/v thymoquinone, from about 2% to about 6% byvolume oil, from about 25% to about 40% by volume surfactant, and water.7. The pharmaceutical composition according to claim 6, wherein thesurfactant comprises Tween-20, the oil comprises Clove Oil, and thepharmaceutical composition is formulated as a microemulsion.
 8. Thepharmaceutical composition according to claim 7, wherein thepharmaceutical composition comprises 0.1% w/v thymoquinone, 3% by volumeclove oil, 30% by volume Tween-20, and the remainder of thepharmaceutical composition comprises water.
 9. The pharmaceuticalcomposition according to claim 6, further comprising at least onesupplemental antimicrobial agent selected from the group consisting ofan aminoglycoside, an ansamycin, a carbapenem, a cephalosporin, aglycopeptide, a lincosamide, a lipopeptide, a macrolide, a monobactam, anitrofuran, an oxazolidinone, a penicillin, a penicillin combination, apolypeptide, a quinolone, a fluoroquinolone, a sulfonamide, atetracycline, an anti-mycobacterial, an antibiotic, an essential oil,the active constituent of an essential oil, myrrh, and an activeingredient of myrrh. 10-16. (canceled)